Khade Rahul L, Adukure Ronald Daisuke, Zhao Xinyi, Wang Carolyn, Fasan Rudi, Zhang Yong
Department of Chemistry and Chemical Biology, Stevens Institute of Technology 1 Castle Point Terrace Hoboken NJ 07030 USA
Department of Chemistry and Biochemistry, University of Texas at Dallas Richardson TX 75080 USA
Catal Sci Technol. 2025 Jan 13;15(6):1802-1813. doi: 10.1039/d4cy00999a. eCollection 2025 Mar 17.
Engineered heme proteins possess excellent biocatalytic carbene N-H insertion abilities for sustainable synthesis, and most of them have His as the Fe axial ligand. However, information on the basic reaction mechanisms is limited, and ground states of heme carbenes involved in the prior computational mechanistic studies are under debate. A comprehensive quantum chemical reaction pathway study was performed for the heme model with a His analogue as the axial ligand and carbene from the widely used precursor ethyl diazoacetate with aniline as the substrate. The ground state of this heme carbene was calculated by the high-level complete active space self-consistent field (CASSCF) approach, which shows a closed-shell singlet that is consistent with many experimental works. Based on this, DFT calculations of ten main reaction pathways were compared. Results showed that the most favorable pathway involved the initial formation of the metal-bound ylide, followed by a concerted rearrangement/dissociation transition state to form the free enol, which then underwent a water-assisted proton transfer process to yield the final N-H insertion product. This computational prediction was validated new experimental data using His-ligated myoglobin variants with different types of carbenes. Overall, this is the first comprehensive computational mechanistic study of heme carbene N-H insertions, particularly for neutral His ligated heme proteins and the first high-level CASSCF confirmation of the ground state of the used heme carbene. The experimental results are also the first in this field. Overall, these results build a solid basis for the proposed reaction mechanism to facilitate future biocatalytic carbene N-H insertion studies.
工程化血红素蛋白在可持续合成方面具有出色的生物催化卡宾N-H插入能力，并且它们中的大多数以组氨酸作为铁的轴向配体。然而，关于基本反应机制的信息有限，并且先前计算机制研究中涉及的血红素卡宾的基态存在争议。我们对以组氨酸类似物作为轴向配体、以广泛使用的前体重氮乙酸乙酯产生的卡宾与苯胺作为底物的血红素模型进行了全面的量子化学反应途径研究。通过高水平的完全活性空间自洽场（CASSCF）方法计算了该血红素卡宾的基态，结果显示其为闭壳单重态，这与许多实验工作一致。基于此，比较了十条主要反应途径的密度泛函理论（DFT）计算结果。结果表明，最有利的途径涉及首先形成金属结合的叶立德，随后是协同重排/解离过渡态以形成游离烯醇，然后该烯醇经历水辅助的质子转移过程以产生最终的N-H插入产物。使用具有不同类型卡宾的组氨酸连接的肌红蛋白变体的新实验数据验证了这一计算预测。总体而言，这是对血红素卡宾N-H插入的首次全面计算机制研究，特别是针对中性组氨酸连接的血红素蛋白，也是对所用血红素卡宾基态的首次高水平CASSCF确认。实验结果在该领域也是首次。总体而言，这些结果为所提出的反应机制奠定了坚实基础，以促进未来生物催化卡宾N-H插入研究。
